Cytokines are proteins released by lymphocytes, endothelial cells, neurons, glia, mononuclear cells and transformed or tumorigenic cells upon contact with specific antigens, proteins or other cells. Generally, they are important cell-cell communication and cellular regulation. More specifically, they affect the function of all cell types involved in the immune and hematopoietic systems. Furthermore, they have been implicated in the pathophysiology of a wide range of diseases. Examples of cytokines with therapeutic importance include interferons a and g, interleukins 1, 2, 3, 4, 6, 10 and 12, tumor necrosis factor a, colony stimulating factors (CSF) including granulocytemacrophage colony stimulating factor (GM-CSF), M-CSF and G-CSF, and erythropoietin.
Cytokines hold considerable promise for anti-cancer therapy. It is accepted that a local inflammatory response accompanied by anti-tumor activity can be induced by cytokines. GM-CSF, interleukins 2, 4, 6, and 12, as well as tumor necrosis factor a (TNF-a) have all shown considerable promise as anti-cancer therapeutics. Ideally, therapeutic cytokines would be induced at high levels only in the vicinity of the tumor. Unfortunately this goal has been impossible to achieve with systemic administration or even local infusion of proteinaceous cytokines. Difficulties with this approach included significant side effects, the great expense of the amounts required and imperfect delivery technology. In addition, cytokines expressed in vivo after gene transfer tend to be poorly expressed, despite their introduction in transcriptionally active expression constructs. The poor expression of cytokines after gene transfer has limited their widespread application in the treatment of cancer patients. The inability of cytokines to be expressed effectively in vivo likely reflects intrinsic cellular and molecular mechanisms which rigorously control cytokine production.
Protooncogenes are normal cellular genes which, upon loss of function, structure or regulation, can induce the conversion of normal cells into cancer cells. The loss of normal regulation typically involves genetic mutation such as chromosomal rearrangement. A variety of leukemias are well characterized for such rearrangements, with the most well known being the Philadelphia chromosome abnormality. In normal cells, protooncogenes regulate critical cell pathways which determine the growth rate, differentiation and cellular function. Classes of molecules known as tumor suppressing genes appear to act as anti-oncogenes by blocking the transforming effects of oncogene overexpression. At this time, the most obvious and best-supported use of protooncogene protein products would be as vaccines to elicit immune responses in patients who harbor tumors induced by oncogene overexpression. Thus, there may also be expanding uses for in vivo protooncogene expression after gene transfer.
A number of studies have demonstrated that post-transcriptional regulation is the dominant means that lymphoid and fibroblastic cells control cytokine expression. In resting cells, mRNAs for cytokines such as GM-CSF, interleukin 2, interleukin 3 or TNF-a are intrinsically unstable with decay rates (T.sub.1/2) on the order of 20-40 minutes. This is primarily due to the presence of adenosine-uridine rich (AU-rich) sequence elements, or "ARE", located in the 3' untranslated region (UTR). For example, these AU-rich elements cause GM-CSF or interleukin 3 mRNAs to be rapidly degraded in the cytoplasm of resting cells. The rapid decay of mRNAs severely reduces mRNA accumulation and hence the amount of protein that can be produced.
The AU-rich elements are composed of multiple reiterations of the pentameric sequence adenosine-uridine-uridine-uridine-adenosine (AUUUA). In GM-CSF mRNA the AUUUA elements are present in tandem array (i.e., AUUUAUUUAUUUA (SEQ ID NO:1)) but this arrangement appears not to be obligatory. Other unstable cytokine mRNAs including interferon gamma, TNF-a and interleukin 6 contain AUUUA motifs which are dispersed throughout the 3' untranslated region. Removal of the AU-rich elements from GM-CSF mRNA led to increased stability of the mutant message while inclusion of the AU-rich element into the 3' untranslated region of the previously stable globin mRNA caused the latter to be rapidly decayed. These and other data have demonstrated that the AU-rich element causes cytokine mRNAs to be rapidly degraded in the cytoplasm of resting cells.
Protooncogene mRNAs often contain AU-rich elements which are identical to those in the cytokine mRNAs. Very little is known about the mechanisms which underlie the control of protooncogene mRNA stability. The inclusion of AU-rich elements in many of these molecules suggests that protooncogene mRNAs will be similarly if not identically regulated as cytokine mRNAs. In two systems, however (fos and myc) the AU-rich element functions in tandem with a poorly described second element located in the coding region. Either element appears sufficient to induce the rapid decay of fos or myc mRNA in resting cells.
Lymphocytes activated by phorbol esters, cytokines including interleukin 1 or TNF-a, or plant lectins (phytohemagglutinin-PHA) show dramatically increased levels of cytokine mRNA. This transcriptional upregulation, however, plays a minimal role in contributing to increased steady state levels of mRNA after cell activation. Instead, mRNA accumulation appears to be determined by a near complete inhibition of cytokine mRNA decay. Therefore, cells contain mechanisms which can attenuate or accelerate the decay of cytokine mRNAs to preserve appropriate cell function. This regulation appears to revolve around the AU-rich element and as discussed later, appears to be mediated by proteins which interact with it.
In an effort to understand how the degradation of mRNAs containing AU-rich elements are regulated, Malter et al. have assayed cytosolic lysates from activated cells for proteins which can bind to the AUUUA motif. They have described a protein factor which specifically interacts with this element and based upon its binding specificity for AUUUA motifs, denoted it the "AU-binding factor" or simply "AUBF." See J. S. Malter, Science, 246, 664-666 (1989); J. S. Malter et al., J Biological Chemistry, 226, 3167-3171 (1991); and P. Gillis et al., J. Biological Chemistry, 266, 3172-3177 (1991). AUBF specifically binds to multiple reiterations of the AUUUA sequence. This factor is not detectable in resting or quiescent cells (cells which have not entered the cell cycle) but it can be rapidly induced after cell activation with phorbol esters, lectins or cytokines. In addition, Malter et al. have detected constitutive AUBF activity in many tumor cell lines including those from lymphoid, fibroblastic or neural origin. Many of these same cell lines contain abnormally stable cytokine mRNAs.
The disruption of the AU-rich elements ("ARE") with a variety of nucleotide substitutions have been uniformly shown to stabilize the resultant cytokine mRNA. In all cases, the more stable mRNA accumulated to higher than wild type levels. However, the accumulated, mutant mRNA has not been shown to be efficiently translated. This has led a variety of investigators to propose that there is an inverse correlation between cytokine mRNA stability and translation. See, for example, Bhonsale et al., Genes and Development, 6, 1927 (1992). Thus, it is believed that the more stable the mRNA, the less it translates, which results in decreased production of the regulatory molecule, e.g., cytokines.
Additional work in which cytokine DNAs have been genetically engineered to be poorly translated by a mutation of the start codon or ribosome binding site, generally produced very stable cytokine mRNAs. This has furthered current thinking that the more stable cytokine mRNAs become, the more poorly translated they are.
Therefore, a need exists for a method to enhance the production of cytokines from stabilized mRNAs in a manner that can enhance the production of cytokines in cells or tissues.